Deoxyribosyl Transfer Catalysis with <i>trans‐N</i>‐Deoxyribosylase

Danzin, Charles; Cardinaud, R.

doi:10.1111/j.1432-1033.1974.tb03763.x

Cited by 28 publications

(11 citation statements)

References 22 publications

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“…The cleavage reaction proceeds through an acyl enzyme intermediate in which an active site glutamate acts as the nucleophile [35,36]. Kinetic evaluation of the enzyme has revealed that nucleoside 2′-deoxyribosyltransferase utilizes a ping-pong kinetic mechanism [37].…”

Section: Discussionmentioning

confidence: 99%

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

Goodenough-Lashua¹,

Garcia²

2003

Bioorganic Chemistry

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AbstracttRNA-guanine transglycosylase (TGT) is a key enzyme in the post-transcriptional modification of certain tRNAs with the pyrrolopyrimidine base queuine. TGT is required for pathogenicity in Shigella flexneri, a human pathogen, and therefore is potentially a novel antibacterial target. Previous work has indicated that the TGT reaction proceeds through a covalent enzyme-tRNA complex [Biochemistry 40 (2001) 14123]. To further substantiate this mechanism, the determination of the kinetic mechanism for the TGT reaction was undertaken. Computational and graphical analyses of initial velocity data are most consistent with a ping-pong kinetic mechanism. The modes of inhibition of 7-methylguanine with respect to both guanine (competitive) and tRNA (uncompetitive) indicate that tRNA binds first to the enzyme. This kinetic mechanism is consistent with the covalent intermediate chemical mechanism and with our earlier study of a mechanism-based inhibitor [7-fluoromethyl -7-deazaguanine, Biochemistry 34 (1995) 15539] in which TGT inactivation was dependent upon the presence of tRNA.

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Section: Discussionmentioning

confidence: 99%

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

Goodenough-Lashua¹,

Garcia²

2003

Bioorganic Chemistry

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“…A kinetic study [18] of trans-N-deoxyribosylase-I established a ping-pong mechanism in which the donor forms a first complex with the enzyme and the first product is released before the second substrate (acceptor) binds to the deoxyribosyl . enzyme.…”

Section: Quaternarj Structurementioning

confidence: 99%

Trans‐N‐Deoxyribosylase: Purification by Affinity Chromatography and Characterization

Holguín¹,

Cardinaud²

1975

European Journal of Biochemistry

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trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively : trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases : trans-N-deoxyribosylase-11. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86000 (f. 4000).In some Lactobacilli a trans-N-deoxyribosylase catalyses the transfer of the deoxyribosyl moiety from one purine or pyrimidine base to another [I]. dRib-Pur + Pur'gdRib-Pur' + Pur (PurePur) dRib-Pur + PyredRib-Pyr + Pur (Pur=Pyr) dRib-Pyr + Pyr'edRib-Pyr' + Pyr (PyrePyr) For short these three transfer reactions will be referred to as (PurePur), (Pur+Pyr) and (PyrePyr) transfers respectively.Early studies of the enzyme from Lactobacillus helveticus [l] have shown that the transfer reaction does not require the presence of phosphate ions. On the other hand Tris was found to have a partial inhibitory action [2]. For a Tris concentration of 0.1 M, (PurGPyr) and (PyrePyr) transfers could be completely inhibited whereas higher concentrations of Tris resulted in a maximum inhibition of 30 % for the (PurePur) transfer. These observations led Roush and Betz to suggest the presence of two distinct proteins : one responsible for the (Pur+Pur) transfer not inhibited by Tris and the other (inhibited by Tris) catalyzing the ( P u r e Pyr) and (PyrePyr) transfers and possibly also the (PurePur) transfer.Abbreviations. Abbreviations follow CBN recommandations, see Eur. J . Biochem. 15, 203-208 (1970): Pur, a purine base; Pyr, a pyrimidine base; (dIno + Ade) denotes a transfer in which deoxyinosine and adenine are the substrates. SE-, sulfoethyl; CM-, carboxymethyl.Enzymes. trans-N-Deoxyribosylase or purine(pyrimidine) nucleoside : purine(pyrimidine) deoxyribosyl transferase (EC 2.4.2.6); xanthine oxidase or xanthine : oxygen oxidoreductase (EC 1.2.3.2).

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“…NDTs from Lactobacillus helveticus and Lactobacillus leichmannii have been well studied (2,25,26,28,29), and their kinetic mechanisms as well as their catalytic and substrate binding sites have been characterized. The transferase reaction proceeds via a ping-pong bi-bi mechanism by formation of a covalent deoxyribosyl enzyme intermediate (3,15,16). Likewise, a glutamyl residue (Glu98) has been proven essential for activity (40,41,46).…”

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confidence: 99%

Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

Fernández‐Lucas

Acebal

Sinisterra³

et al. 2010

Appl Environ Microbiol

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A novel type II nucleoside 2-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% ␣-helix, 10% ␤-strand, 16% ␤-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T m ) of 64°C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40°C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2-fluorodeoxyribonucleosides was carried out.Nucleoside 2Ј-deoxyribosyltransferases (NDTs) (EC 2.4.2.6) catalyze the exchange between the purine or pyrimidine base of 2Ј-deoxyribonucleosides and free pyrimidine or purine bases (10, 25). These enzymes are specific for 2Ј-deoxyribonucleosides, regioselective (N-1 glycosylation in pyrimidine and N-9 in purine), and stereoselective (␤-anomers are exclusively formed) (26) (Fig. 1).Deoxyribosyltransferases are classified into two classes depending on their substrate specificity: type I (NDT I), specific for purines (Pur 7 Pur), and type II (NDT II), which catalyzes the transfer between purines and/or pyrimidines (Pur 7 Pur, Pur 7 Pyr, Pyr 7 Pyr) (10, 25). These enzymes were initially described for lactobacilli (27,28), and they are involved in the nucleoside salvage pathway for DNA synthesis (23), although this remains unclear in Lactococcus lactis subsp. lactis (36). NDTs have been also found in some species of Streptococcus (11), in parasitic unicellular eukaryotic organisms such as Crithidia luciliae (49, 50), in Trypanosoma brucei (6), and in Borrelia burgdorferi (33). NDTs from Lactobacillus helveticus and Lactobacillus leichmannii have been well studied (2,25,26,28,29), and their kinetic mechanisms as well as their catalytic and substrate binding sites have been characterized. The transferase reaction proceeds via a ping-pong bi-bi mechanism by formation of a covalent deoxyribosyl enzyme intermediate (3,15,16). Likewise, a glutamyl residue (Glu98) has been proven essential for activity (40,41,46). Enzymatic natural and nonnatural nucleoside synthesis in a one-pot reaction by NDTs provides an interesting alternative to traditional multistep chemical methods (13,34). Indeed, chemical glycosylation includes several protection-deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Whereas previously described NDTs accept different nucleosides fr...

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Deoxyribosyl Transfer Catalysis with trans‐N‐Deoxyribosylase

Cited by 28 publications

References 22 publications

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

Trans‐N‐Deoxyribosylase: Purification by Affinity Chromatography and Characterization

Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

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